PROJECT SUMMARY The proper regulation of gene expression by bacteria requires many interacting factors operating at multiple points, including at the transcriptional, post-transcriptional, and post-translational levels. Numerous bacteria co- ordinate gene expression programs via the activity of global transcriptional regulators, including the catabolite repressor protein Crp. Crp links nutritional status (typically in the form of carbon source availability such as glucose levels) with gene expression, and in some bacterial pathogens, this global regulator Crp not only activates metabolic and housekeeping genes but also promotes the synthesis of virulence factors. Crp influences gene expression through the transcription of both protein-coding genes as well as genes encoding small, noncoding RNAs (sRNAs), linking Crp to post-transcriptional mechanisms of gene regulation. Our group recently reported that Crp is regulated at the post-transcriptional level via the activity of the small RNA chaperone Hfq; specifically, we found that at 37C Y. pestis stimulates the synthesis of Crp protein levels in an Hfq-dependent manner, and this activity occurs specifically at the crp 5' untranslated region (UTR). Indeed, unlinking Crp synthesis from Hfq restores the expression of the plasminogen activator protease Pla, an essential Y. pestis virulence factor that is directly regulated transcriptionally by Crp. Recent reports have demonstrated that the E. coli Crp protein is also regulated at the post-transcriptional level via Hfq; this suggests that the novel mechanisms of Crp regulation we have identified with our studies of Y. pestis may be broadly applicable to other Gram-negative bacteria. In support of our analyses, unlinking Crp synthesis from Hfq restores the expression of Pla, and the impact of this additional post-transcriptional mechanism of fine- tuning Crp levels enhances the infectious process during pneumonic plague. However, the specific genetic factors and the regulatory mechanisms by which Y. pestis regulates Crp at the post-transcriptional level are still not known. Therefore, we propose in this study to identify the genetic elements, including sRNAs, that contribute to this post-transcriptional regulation of Crp specifically at the crp 5' UTR by using a high-throughput, saturating transposon mutagenesis screen. We also propose to examine the specific post-transcriptional effects of Crp regulation on virulence during experimental bubonic and pneumonic plague. We expect that these studies will expand our understanding of how and why Y. pestis regulates the global transcription factor Crp at the post-transcriptional level, which we predict will also impact our general knowledge of how other bacteria such as E. coli also control Crp synthesis as well.